The ultimate objective of the proposed research is to elucidate mechanisms involved in the regulation of gene transcription in eukaryotic cells during cellular growth and differentiation. The proposed studies will focus on the wheat embryo system which undergoes a dramatic, abrupt activation of messenger RNA synthesis and a gradual activation of ribosomal RNA transcription during germination. The ease by which unlimited quantities of tissue can be caused to undergo this activation synchronously should allow biochemical analysis of transcriptional components during changing rates of in vivo RNA synthesis. Specifically, the multiple nuclear RNA polymerases will be purified during germination and their intracellur concentrations, endogenous nuclear activities, chemical modifications, structural alterations, compartmentalization and interaction with other nuclear enzymes or components, will be correlated with activation or modulation of in vivo rates of RNA synthesis. Analytical methods will involve affinity chromatography of cellular extracts on agarose columns containing RNA polymerase I-, RNA polymerase II- and RNA polymerase III-specific immunoglobulins and subsequent analysis of purified polymerases by two-dimensional gel electrophoresis. This latter technique will be coupled with autoradiographic analysis in those instances where in vitro labelling will be done to monitor subunit synthesis, turnover or modification. Also, in vitro nuclear systems will be developed which are capable of RNA chain initiation by endogenous and exogenously added purified RNA polymerases I, II and III, and an assay developed for possible transcription factors which regulate the activities of specific RNA polymerases and hence specific genes.